Laminins are a family of basement membrane proteins which function in cell differentiation, adhesion, and migration, in addition to being true structural components (Tryggvason K, Curr. Opn. Cell Biol., 1993, 5:877-882, this and all following references are hereby incorporated by reference). The laminin molecule is a cross-shaped heterotrimer consisting of one heavy chain (≈400 kd) and two light chains, β and γ (130-200 kd) (nomenclature according to Burgeson et al., Matrix Biol., 1994, 14:209-211). Laminins exist as several isoforms each having a unique combination of α, β and γ chains. Thus far, ten genetically distinct laminin chains, α1-α5, β1-β3 and γ1-γ2 are known.
In the laminin molecule the three chains are associated through a carboxyl terminal coiled coil (long arm), most of the chains having a free amino terminal short arm. Additionally, all the α chains have a large globular G domain at the carboxyl terminus. Laminin can contribute to the structural framework of the basement membrane, but it is also believed to have a role in cell differentiation, proliferation, adhesion and migration (Timpl, R. & Brown, J. C. (1994) Matrix Biol. 14: 275-81, Yurchenco, P. D. & O'Rear, J. J. (1994) Curr. Opin. Cell. Biol. 6: 674-81). Many of the laminin chains have tissue- and cell-specific distribution which may vary between different developmental stages, indicating specific functions for the various chains and isoforms. Evidence for tissue-specific roles of some of the laminin chains has come from identification of mutations in the α2 chain gene in muscular dystrophies in mouse and man (Xu, H., Wu, X. R., Wewer, U. M. & Engvall, E. (1994) Nature Genet. 8: 297-301; Heibling-Leclerc, A., Zhang, X., Topaloglu, H., Cruaud, C., Tesson, F., Weissenbach, J., Tome', F., Schwartz, K., Fardeau, M., Tryggvason, K. & Guicheney, P. (1995) Nature Genet. 11: 216-218; Nissinen, M., Heibling-Leclerc, A., Zhang, X., Evangelista, T., Topaloglu, H., Cruaud, C., Weissenbach, J., Fardeau, M., Tome', F. M. S., Schwartz, K., Tryggvason, k. & Guicheney, P. (1996) Am. J. Hum. Genet. 58: 1177-1184), as well as in the genes for the α3, β3 and γ2 chains in epidermolysis bullosa (Pulkkinen, L., Christiano, A. M., Airenne, T., Haakana, H., Tryggvason, K. & Uitto, J. (1994a) Nature Genet. 6: 293-297; Pulkkinen, L., Christiano, A. M., Gerecke, D., Wagman, D. W., Burgeson, R. E., Pittelkow, M. R. & Uitto, J. (1994b) Genomics 24: 357-60; Aberdam, D., Galliano, M. F., Vailly, J., Pulkkinen, L., Bonifas, J., Christiano, A. M., Tryggvason, K., Uitto, J., Epstein, E. J., Ortonne, J. P. & Meneguzzi, G. (1994) Nature Genet. 6: 299-304; Kivirikko, S., McGrath, J. A., Baudoin, C., Aberdam, D., Ciatti, S., Dunnill, M. G. S., McMillan, J. R., Eady, R. A. J., Ortonne, J-P., Meneguzzi, G., Uitto, J. & Christiano, A. M. (1995) Hum. Mol. Genet. 4: 959-962; Vidal, F., Baudoin, C., Miquel, C., Galliano, M-F., Christiano, A. M., Uitto, J., Ortonne, J-P. & Meneguzzi, G. (1995) Genomics 30: 273-280).
Laminin-5, is a unique subepithelial basement membrane isoform with the molecular formula α3:β3:γ2 chains (Burgeson, R. E., Chiquet, M., Deutzmann, R., Ekblom, P., Engel, J., Kleinman, H., Martin, G. R., Meneguzzi, G., Paulsson, M., Sanes, J., Timpl, R., Tryggvason, K., Yamada, Y., & Yurchenco, P. D. (1994) Matrix Biol. 14: 209-211). Determination of the primary structure of the human α3, β3 and γ2 chains has revealed that all these chains are truncated in the short arm relative to the corresponding chains of laminin-1 (Kallunki, P., Sainio, K., Eddy, R., Byers, M., Kallunki, T., Sariola, H., Beck, K., Hirvonen, H., Shows, T. B. & Tryggvason, K. (1992) J. Cell Biol. 119: 679-693; Ryan, M. C., Tizard, R., VanDevanter, D. R. & Carter, W. G. (1994) J. Biol. Chem. 269: 22779-22787; Gerecke, D. R., Wagman, D. W., Champliaud, M. F. & Burgeson, R. E. (1994) J. Biol. Chem). Additionally, the γ2 chain exists in two forms differing in the length of their carboxyl terminal end due to alternative splicing Kallunki, P., Sainio, K., Eddy, R., Byers, M., Kallunki, T., Sariola, H., Beck, K., Hirvonen, H., Shows, T. B. & Tryggvason, K. (1992) J. Cell Biol. 119: 679-693; Ryan, M. C., Tizard, R., VanDevanter, D. R. & Carter, W. G. (1994) J. Biol. Chem. 269: 22779-22787; Gerecke, D. R., Wagman, D. W., Champliaud, M. F. & Burgeson, R. E. (1994) J. Biol. Chem; Airenne, T., Haakana, H., Sainio, K., Kallunki, T., Kallunki, P., Sariola, H. & Tryggvason, K. (1996) Genomics 32: 54-64). Immunolocalization of the laminin-5 protein (previously termed kalinin, nicein or epiligrin) to anchoring filaments (Verrando, P., Hsi, B., Yeh, C., Pisani, A., Serieys, N., & Ortonne, J. (1987) Exp. Cell Res. 170:116-128; Carter, W. G., Ryan, M. C. & Gahr, P. J. (1991) Cell 65: 599-610; Rousselle, P., Lunstrum, G. P., Keene, D. R. & Burgeson, R. E. (1991) J. Cell Biol, 114: 567-576) as well as epithelium-specific expression of the γ2 chain (Kallunki, P., Sainio, K., Eddy, R., Byers, M., Kallunki, T., Sariola, H., Beck, K., Hirvonen, H., Shows, T. B. & Tryggvason, K. (1992) J. Cell Biol. 119: 679-693) already implied its role as an epithelial attachment component. The adhesion properties of laminin-5 have been demonstrated in several cell attachment studies (Carter, W. G., Ryan, M. C. & Gahr, P. J. (1991) Cell 65: 599-610; Rousselle, P., Lunstrum, G. P., Keene, D. R. & Burgeson, R. E. (1991) J. Cell Biol. 114: 567-576; Sonnenberg, A., Calafat, J., Janssen, H., Daams, H., van der Raaij-Helmer, L. M. H., Falcioni, R., Kennel, S. J., Aplin, J. D., Baker, J., Loizidou, M. & Garrod, D. (1991) J. Cell Biol. 113: 907-917; Niessen, C. M., Hogervorst, F., Jaspars, L. H., De Melker, A. A., Delwel, G. O., Hulsman, E. H., Kuikman, I. & Sonnenberg, A. (1994) Exp. Cell. Res. 211: 360-367; Rousselle, P. & Aumailley, M. (1994) J. Cell Biol. 125:205-214). The adhesive function of laminin-5 has been shown to be mediated through α3β1 and α6β4 integrins (Carter, W. G., Ryan, M. C. & Gahr, P. J. (1991) Cell 65: 599-610; Sonnenberg, A., Calafat, J., Janssen, H., Daams, H., van der Raaij-Helmer, L. M. H., Falcioni, R., Kennel, S. J., Aplin, J. D., Baker, J., Loizidou, M. & Garrod, D. (1991) J. Cell Biol. 113: 907-917; Rousselle, P. & Aumailley, M. (1994) J. Cell Biol. 125:205-214). Direct evidence for the crucial role of laminin-5 for epithelial cell attachment has come from the identification of mutations in the genes of all the subunit chains (Pulkkinen, L., Christiano, A. M., Airenne, T., Haakana, H., Tryggvason, K. & Uitto, J. (1994) Nature Genet. 6: 293-297; Pulkkinen, L., Christiano, A. M., Gerecke, D., Wagman, D. W., Burgeson, R. E., Pittelkow, M. R. & Uitto, J. (1994b) Genomics 24: 357-60; Aberdam, D., Galliano, M. F., Vailly, J., Pulkkinen, L., Bonifas, J., Christiano, A. M., Tryggvason, K., Uitto, J., Epstein, E. J., Ortonne, J. P. & Meneguzzi, G. (1994) Nature Genet. 6: 299-304; Kivirikko, S., McGrath, J. A., Baudoin, C., Aberdam, D., Ciatti, S., Dunnill, M. G. S., McMillan, J. R., Eady, R. A. J., Ortonne, J-P., Meneguzzi, G., Uitto, J. & Christiano, A. M. (1995) Hum. Mol. Genet. 4: 959-962; Vidal, F., Baudoin, C., Miquel, C., Galliano, M-F., Christiano, A. M., Uitto, J., Ortonne, J-P. & Meneguzzi, G. (1995) Genomics 30: 273-280) in the Herlitz's variant of junctional epidermolysis bullosa, a lethal skin blistering disease caused by disruption of the epidermal-dermal junction. One and possibly the only cell adhesion site of laminin-5 has been localized to the long arm (Rousselle, P. & Aumailley, M. (1994) J. Cell Biol. 125:205-214; Rousselle, P., Golbik, R., van der Rest, M. & Aumailley, M. (1995) J. Biol. Chem. 270:13766-13770). However, a mutation in one junctional epidermolysis bullosa patient causing an in-frame deletion of 73 residues from domains III and IV of the short arm of the laminin γ2 chain indicates a role for this part of the chain for the anchorage of epithelial cells to the extracellular matrix (Pulkkinen, L., Christiano, A. M., Airenne, T., Haakana, H., Tryggvason, K. & Uitto, J. (1994) Nature Genet. 6: 293-297).
By in situ hybridization the γ-2 chain was found to be expressed in epithelial cells of many embryonic tissues such as those of skin, lung, and kidney (Kallunki et al., 1992, supra.), and antibodies to kalinin/laminin 5, react with basement membranes of the same tissues (Rousselle et al., 1991, supra.; Verrando et al., Lab. Invest., 1991, 64:85-92).
The different laminin chains have been shown to have quite varying tissue distribution as determined by immunohistological studies, Northern, and in situ hybridization analyses. For example, the A and M chains on the one hand, and the B1 (β-1) and S (β-2) chains on the other, have been shown to be mutually exclusive (see for example Vuolteenaho et al., J. Cell Biol., 1994, 124:381-394). In vitro studies have indicated that laminin mediates a variety of biological functions such as stimulation of cell proliferation, cell adhesion, differentiation, and neurite outgrowth. The cellular activities are thought to be mediated by cell memebrane receptors, many of which are members of the integrin family (Ruoslahti, E. J. Clin. Invest., 1991, 87:1-5; Mecham, R. P. FASEB J., 1991, 5:2538-2546; Hynes, R. Cell, 1992, 69:11-25). Recently a new nomenclature for describing laminins has been agreed to as in the following Table 1 (after Burgeson et al., 1994, supra.):
TABLE 1Laminin Chains and GenesNewPreviousGeneα1A, AeLAMA1α2M, AmLAMA2α3200 kDaLAMA3β1B1, B1eLAMB1β2S, B1sLAMB2β3140 kDaLAMB3γ1B2, B2eLAMC1γ2B2tLAMC2Heterotrimers of LamininNewChainsPreviousLaminin-1α1β1γ1EHS lamininLaminin-2α2β1γ1merosinLaminin-3α1β2γ1s-lamininLaminin-4α2β2γ1s-merosinLaminin-5α3β3γ2kalinin/niceinLaminin-6α3β1γ1k-lamininLaminin-7α3β2γ1ks-laminin
Cell migration is one of the biological functions proposed for laminin (Timpl, R. & Brown, J. C. (1994) Matrix Biol. 14: 275-81). Cellular movement is required for various physiological and pathological processes, such as during embryogenesis, wound healing, angiogenesis and tumor invasion. Immunohistochemical and in situ hybridization studies have shown induction of laminin-5 expression in migrating keratinocytes during wound healing (Ryan, M. C., Tizard, R., VanDevanter, D. R. & Carter, W. G. (1994) J. Biol. Chem. 269: 22779-22787; Larjava, H., Salo, T., Haapasalmi, K., Kramer, R. H. & Heino J. (1993) Clin. Invest. 92: 1425-1435; Pyke, C., Romer, J., Kallunki, P., Lund, L. R., Ralfkiaer, E., Dano, K. & Tryggvason, K (1994) Am. J. Pathol. 145: 782-791). The γ2 chain of laminin-5 has also been shown to be strongly expressed in malignant cells located at the invasion front of several human carcinomas, as determined by in situ hybridization and immunohistochemical staining (Pyke, C., Romer, J., Kallunki, P., Lund, L. R., Ralfkiaer, E., Dano, K. & Tryggvason, K (1994) Am. J. Pathol. 145: 782-791; Pyke, C., Salo, S., Ralfkiaer, E., Romer, J., Dano, K. & Tryggvason, K. (1995) Cancer Res. 55: 4132-4139). Since laminin-1 has been found to inhibit keratinocyte migration in vitro (Woodley, D. T., Bachmann, P. M. & O'Keefe, E. J. (1988) J. Cell. Physiol. 136: 140-146), and as the laminin α1, β1 and γ1 chains are only weakly expressed throughout cancerous areas with no apparent correlation to sites of invasion, laminin-5 has been proposed to have a role in the migration event (Pyke, C., Romer, J., Kallunki, P., Lund, L. R., Ralfkiaer, E., Dano, K. & Tryggvason, K (1994) Am. J. Pathol. 145: 782-791).